Methods of treating retinal disorders with anti-apelin antibodies

ABSTRACT

This disclosure relates to apelin antigen-binding proteins and methods of using the apelin antigen-binding proteins. The antigen-binding protein may comprise an antibody to apelin and can be used to treat pathological conditions involving angiogenesis. The pathological conditions can comprise cancer or retinopathy and/or retinopathy-related complications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/233,364, filed Mar. 26, 2014, which is a national phase entry ofInternational Application No. PCT/US2012/047048, filed Jul. 17, 2012,which claims the benefit of U.S. Provisional Application No. 61/508,862,filed Jul. 18, 2011, all of which are hereby incorporated by referencein their entireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“A-1633-US-CNT_ST25.txt” and was created on Aug. 28, 2015. The text fileis 12,062 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates to apelin antigen-binding proteins and methodsof using the apelin antigen-binding proteins. The antigen-bindingprotein may comprise an antibody to apelin.

BACKGROUND OF THE INVENTION

Apelin is an endogenous ligand for the angiotensin-1-like receptor APJ.Apelin is derived from a 77 amino acid precursor and processed intoseveral active molecular forms including apelin-12, apelin-13, apelin-17and apelin-36. Apelin is expressed in various organs, e.g. heart, lung,kidney, liver and other tissues. Apelin and the apelin receptor (APJ)are a G-protein coupled receptor system that has been found to beinvolved in pathological conditions involving angiogenesis, includingcancer. Recently, apelin has been reported to be involved inhypoxia-induced retinal angiogenesis (Kasai et al. Arterioscler ThrombVasc Biol 2010: 30, 2182-2187). It has also been reported that certaincompositions may inhibit angiogenesis by inhibiting the apelin/APJpathway (U.S. Pat. No. 7,736,646).

Apelin knockout mice have been shown to have impaired retinalvascularization and ocular development (Kasai et al. Arterioscler ThrombVasc Biol 2008: 28, 1717-1722). The impaired vascular developmentoccurred at least in the early postnatal period. Furthermore, it wassuggested that apelin/APJ signaling was involved in a cooperative mannerwith VEGF or FGF2 in this condition.

It has also been reported that vitreous concentrations of apelin weresignificantly higher in patients having proliferative diabeticretinopathy (Tao et al., Invest Opthamol Visual Science 2010: 51,4237-4242).

SUMMARY OF THE INVENTION

The invention relates to antigen-binding proteins that bind to apelinand fragments thereof. In various embodiments the antigen-bindingproteins are antibodies. Additionally, uses are provided for theantigen-binding proteins described herein.

In various embodiments the isolated antigen-binding protein comprises alight chain (SEQ ID NO: 2) and a heavy chain (SEQ ID NO: 27). In otherembodiments, the antigen-binding protein can comprise a polypeptide ofonly SEQ ID NO: 2 or SEQ ID NO: 27. In other aspects the antigen-bindingprotein sequence can be about 95%, about 96%, about 97%, about 98%,about 99% identical to SEQ ID NOs: 2 or 27. The antigen-binding proteincan be a monoclonal antibody or fragment thereof. In certainembodiments, the antibody can be a mouse antibody, a humanized antibody,a chimeric antibody, a multispecific antibody, or fragment of a mouseantibody, a humanized antibody, a chimeric antibody or a multispecificantibody. In various embodiments, the antigen-binding proteinspecifically binds to an apelin polypeptide comprising the sequenceQRPRLSHKGPMPF (SEQ ID NO: 31) or PRLSHKG (SEQ ID NO: 32) or afull-length human (SEQ ID NO: 3), mouse (SEQ ID NO: 4), rat (SEQ ID NO:5) or bovine sequence (SEQ ID NO: 6).

In yet other embodiments the antigen-binding protein can have a K_(D) of6 nM or less. The antigen binding protein can also be a neutralizingantibody. Other embodiments can comprise a nucleic acid sequenceencoding any of the above antigen-binding proteins. In variousembodiments, the antigen binding protein can inhibit angiogenesis. Theangiogenesis can be inhibited in cancer or diabetic retinopathy and/orretinopathy-associated complications. Inhibition of angiogenesis can bemeasured in vitro by decreased length during tube formation.

In various embodiments the isolated antigen-binding protein comprises apolypeptide having the sequences of SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. In someembodiments the isolated antigen binding protein can comprise only one,two, three, four or five of the SEQ ID NOs: 23-30. In other aspects theantigen-binding protein sequence can be about 95%, about 96%, about 97%,about 98%, about 99% identical to SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. Theantigen-binding protein can be a monoclonal antibody or fragmentthereof. In certain embodiments, the antibody can be a mouse antibody, ahumanized antibody, a chimeric antibody, a multispecific antibody, orfragment of a mouse antibody, a humanized antibody, a chimeric antibodyor a multispecific antibody. In various embodiments, the antigen-bindingprotein specifically binds to a polypeptide comprising the sequenceQRPRLSHKGPMPF (SEQ ID NO: 31) or PRLSHKG (SEQ ID NO: 32) or afull-length human, mouse, rat or bovine sequence (SEQ ID NOs: 3-6).

In yet other embodiments the antigen-binding can have a K_(D) of 6 nM orless. The antigen binding protein can also be a neutralizing antibody.Other embodiments can comprise a nucleic acid sequence encoding any ofthe above antigen-binding proteins. In various embodiments, the antigenbinding protein can inhibit angiogenesis. The angiogenesis can beinhibited in diabetic retinopathy or cancer. The inhibition ofangiogenesis can be measured in vitro by decreased length during tubeformation.

In various embodiments the invention relates to an expression vectorcomprising a nucleic acid encoding any of the above antigen-bindingproteins. The expression vector can be placed in a host cell. The hostcell can comprise the nucleic acid and the host cell can be a eukaryoticor prokaryotic cell. In various embodiments the eukaryotic host cell canbe a mammalian cell. In certain embodiments a method of producing anantibody is provided. The method can comprise culturing the host cellunder suitable conditions such that the nucleic acid is expressed toproduce the antibody. The antibody can then be recovered from theculture of the host cell.

In various embodiments, a pharmaceutical composition is providedcomprising any of the above antigen-binding proteins and apharmaceutically acceptable carrier, diluent or excipient.

In yet other embodiments a method for detecting the presence of apelinin a biological sample is provided. The method can comprise incubatingthe sample with at least one of the antigen-binding protein set forthabove under conditions that allow binding of the antigen-binding proteinto apelin, and detecting the bound antigen-binding protein or apelin. Invarious embodiments a method of treating a disorder associated withincreased angiogenesis in a mammal in need of treatment is provided. Themethod can comprise administering any of the above antigen-bindingproteins or the above pharmaceutical composition to the mammal. Thedisorder can comprise hemangioma, solid tumors, leukemias, lymphomas,myelomas, plaque neovascularization, corneal diseases, rubeosis,neovascular glaucoma, retinopathy, exudative age-related maculardegeneration (AMD), proliferative diabetic retinopathy (PDR), diabeticmacular edema (DME), neovascular glaucoma, corneal neovascularization(trachoma), pterygium, diabetic retinopathy, retrolental fibroplasia,diabetic neovascularization, macular degeneration, uterine bleeding,endometrial hyperplasia and carcinoma, endometriosis, myometrialfibroids (uterine leiomyomas) and adenomyosis, ovarian hyperstimulationsyndrome, tumorigenesis or cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. This figure presents the sequence of the light chain and heavychain of antibody A (Ab A) to apelin. The CDR regions in the heavy andlight chains are shown in bold, italic type. The signal sequences areunderlined. Additionally, the individual CDR's are presented.

FIGS. 2A-B. These figures provide the individual apelin peptides (FIG.2A) that were tested against Ab A and also show an anti-apelin mAb ELISAof the antibody and the apelin peptides (FIG. 2B)

FIG. 3. This figure presents the results of a neutralization assay withapelin and Ab A.

FIGS. 4A-B. These figures present results of an anti-angiogenesisexperiment. FIG. 4A demonstrates the effect of apelin on tube formationwhich is an indication of angiogenesis. FIG. 4B demonstrates inhibitionof angiogenesis when Ab A is added with apelin.

FIG. 5. This figure presents the full-length sequence of mouse, rat,human and bovine apelin. The NP059109 human sequence is SEQ ID NO: 3.The NP038940 mouse sequence is SEQ ID NO: 4. The NP113800 rat sequenceis SEQ ID NO: 5. The NP776928 bovine sequence is SEQ ID NO: 6.

FIG. 6. This figure demonstrates the effects of systemic administrationof apelin antibody on retinal neovascularization.

FIG. 7. This figure demonstrates the effect of intravitreal injection ofapelin antibody on retinal neovascularization.

DETAILED DESCRIPTION

Apelin antigen-binding proteins (such as antibodies and functionalbinding fragments thereof) that bind to apelin are disclosed herein. Insome embodiments, the antigen-binding proteins bind to apelin andprevent apelin from functioning in various ways. For example, the apelinbinding proteins may inhibit angiogenesis and more specifically, theangiogenesis associated with cancer, retinopathy and/orretinopathy-related complications. The retinopathy can be diabeticretinopathy. Alternatively, the apelin binding proteins may inhibitangiogenesis in tumors or other forms of cancer.

The foregoing summary is not intended to define every aspect orembodiment of the invention, and additional aspects may be described inother sections. The entire document is intended to be related as aunified disclosure, and it should be understood that all combinations offeatures described herein may be contemplated, even if the combinationof features is not found together in the same sentence, or paragraph, orsection of this document.

In addition to the foregoing, as an additional aspect, all embodimentsnarrower in scope in any way than the variations defined by specificparagraphs herein can be included in this disclosure. For example,certain aspects are described as a genus, and it should be understoodthat every member of a genus can be, individually, an embodiment. Also,aspects described as a genus or selecting a member of a genus should beunderstood to embrace combinations of two or more members of the genus.It should also be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment may also be described using“consisting of” or “consisting essentially of” language.

It will be understood that the descriptions herein are exemplary andexplanatory only and are not restrictive of the invention as claimed. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Also, the use of the term “portion” can include partof a moiety or the entire moiety.

It should also be understood that when describing a range of values, thecharacteristic being described could be an individual value found withinthe range. For example, “a pH from about pH 4 to about pH 6,” could be,but is not limited to, pH 4, 4.2, 4.6, 5.1 5.5 etc. and any value inbetween such values. Additionally, “a pH from about pH 4 to about pH 6,”should not be construed to mean that the pH in question varies 2 pHunits from pH 4 to pH 6, but rather a value may be picked from within atwo pH range for the pH of the solution.

In some embodiments, when the term “about” is used, it means the recitednumber plus or minus 5%, 10%, 15% or more of that recited number. Theactual variation intended is determinable from the context.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. As utilized in accordance with thedisclosure, the following terms, unless otherwise indicated, shall beunderstood to have the following meanings:

An antibody or antigen-binding fragment can be an agonist or anantagonist.

An “agonist” refers to an agent that binds to a polypeptide (such as areceptor), or a polynucleotide and stimulates, increases, activates,facilitates, enhances activation, sensitizes or up regulates theactivity or expression of the polypeptide or polynucleotide.

An “antagonist” refers to an agent that inhibits expression of apolypeptide or polynucleotide or binds to, partially or totally blocksstimulation, decreases, prevents, delays activation, inactivates,desensitizes, or down regulates the activity of the polypeptide orpolynucleotide.

“Angiogenesis” should be understood to mean the growth of new bloodvessels. Angiogenesis may occur under conditions of normal or abnormalgrowth (e.g tumor growth). Angiogenesis may also be referred to asvasculogenesis or arteriogenesis

Inhibition of angiogenesis should be understood to occur whenadministration of an angiogenesis inhibitor (e.g. an anti-apelinantibody) is administered in vivo or provided to an in vitro assaysystem and there is a decrease in blood vessels or an indicator of bloodvessel growth. In an in vitro system, the inhibition may be reflected asa decrease in the length of a structure called a “tube” structure. Forexample, an in vitro angiogenesis assay can be measured using CellplayerGFP AngioKit-96 (Essen Bioscience). The assay measures both positive andnegative effects on tube (vessel) formation during angiogenesis whenhuman endothelial cells are co-cultures with other human cells.

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers. Nucleotidescomprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotides. In some embodiments, oligonucleotides are about 10 toabout 60 bases in length. In other embodiments, oligonucleotides areabout 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, or about 20 to about 40 nucleotides in length.Oligonucleotides can be single stranded or double stranded, e.g., foruse in the construction of a mutant gene. Oligonucleotides can be senseor antisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides can be used, for example,as PCR primers, cloning primers or hybridization probes.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences can include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or can include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or can include vector sequences.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences;” sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences.”

An isolated nucleic acid can encode antigen-binding proteins disclosedin various embodiments herein, e.g. an apelin antigen-binding protein oranti-apelin antibody. The nucleic acid is said to be “operably linked”when it is placed into a functional relationship with another nucleicacid sequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are near each other, and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

The term “amino acid” refers to natural and/or non-naturally occurringamino acids, and includes its normal meaning in the art.

The terms “polypeptide” or “protein” means a macromolecule having theamino acid sequence of a native protein, i.e., a protein produced by anaturally-occurring and non-recombinant cell; or the protein can beproduced by a genetically-engineered or recombinant cell, and comprisemolecules having the amino acid sequence of the native protein, ormolecules having deletions from, additions to, and/or substitutions ofone or more amino acids of the native sequence. The term also includesamino acid polymers in which one or more amino acids are chemicalanalogs of a corresponding naturally-occurring amino acid and polymers.The terms “polypeptide” and “protein” specifically encompass inter alia,apelin antigen-binding proteins, antibodies, or sequences that havedeletions from, additions to, and/or substitutions of one or more aminoacid of antigen-binding protein. The term “polypeptide fragment” refersto a polypeptide that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion as compared withthe full-length native protein. Such fragments can also contain modifiedamino acids as compared with the native protein. In various embodiments,fragments can be about five to about 500 amino acids long. For example,fragments can be at least about 5, about 6, about 8, about 10, about 14,about 20, about 50, about 70, about 100, about 150, about 200, about250, about 300, about 350, about about 400, or about 450 amino acidslong. Useful polypeptide fragments include immunologically functionalfragments of antibodies, including binding domains. In the case of anapelin-binding antibody, useful fragments include but are not limited toa CDR region, a variable domain of a heavy and/or light chain, a portionof an antibody chain or just its variable region including one, two,three, four, five or six CDRs, and the like.

The term “isolated protein” means that a subject protein (1) is free ofat least some other proteins with which it would normally be found, (2)is essentially free of other proteins from the same source, e.g., fromthe same species, (3) is expressed by a cell from a different species,(4) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis associated in nature, (5) is operably associated (by covalent ornon-covalent interaction) with a polypeptide with which it is notassociated in nature, or (6) does not occur in nature. Typically, an“isolated protein” constitutes at least about 5%, at least about 10%, atleast about 25%, or at least about 50%, at least about 75%, at leastabout 90% or more of a given sample. Genomic DNA, cDNA, mRNA or otherRNA, of synthetic origin, or any combination thereof can encode such anisolated protein. In various embodiments, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antigen-binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins.

As used herein, the twenty conventional (e.g., naturally occurring)amino acids and their abbreviations follow conventional usage. SeeImmunology—A Synthesis (2nd Ed., E. S. Golub & D. R. Gren, Eds., SinauerAssoc., Sunderland, Mass. (1991)), which is incorporated herein byreference for any purpose. Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids can also be suitable components forpolypeptides of various embodiments described herein. Examples ofunconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

Conservative amino acid substitutions can encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues can be divided into classes based on commonside chain properties:

-   -   Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   Acidic: Asp, Glu;    -   Basic: His, Lys, Arg;    -   Residues that influence chain orientation: Gly, Pro; and    -   Aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions can involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues can be introduced, for example, into regions of ahuman antibody that are homologous with non-human antibodies, or intothe non-homologous regions of the molecule.

In making changes to an antigen-binding protein (such as an antibody),according to certain embodiments, the hydropathic index of amino acidscan be considered. Each amino acid has been assigned a hydropathic indexon the basis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments. Incertain embodiments, the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, i.e., with abiological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One can also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

The term “derivative” refers to a molecule that includes a chemicalmodification other than an insertion, deletion, or substitution of aminoacids (or nucleic acids). In certain embodiments, derivatives comprisecovalent modifications, including, but not limited to, chemical bondingwith polymers, lipids, or other organic or inorganic moieties. Incertain embodiments, a chemically modified antigen-binding protein canhave a greater circulating half-life than an antigen-binding proteinthat is not chemically modified. In certain embodiments, a chemicallymodified antigen-binding protein can have improved targeting capacityfor desired cells, tissues, and/or organs. In some embodiments, aderivative antigen-binding protein is covalently modified to include oneor more water soluble polymer attachments, including, but not limitedto, polyethylene glycol, polyoxyethylene glycol, or polypropyleneglycol. See e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144,4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a derivativeantigen-binding protein comprises one or more polymer, including, butnot limited to, monomethoxy-polyethylene glycol, dextran, cellulose, orother carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of suchpolymers.

In certain embodiments, a derivative is covalently modified withpolyethylene glycol (PEG) subunits. In certain embodiments, one or morewater-soluble polymer is bonded at one or more specific position, forexample at the amino terminus, of a derivative. In certain embodiments,one or more water-soluble polymer is randomly attached to one or moreside chains of a derivative. In certain embodiments, PEG is used toimprove the therapeutic capacity for an antigen-binding protein. Incertain embodiments, PEG is used to improve the therapeutic capacity fora humanized antibody. Certain such methods are discussed, for example,in U.S. Pat. No. 6,133,426, which is hereby incorporated by referencefor any purpose.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J., Adv. Drug Res., 15:29(1986); Veber & Freidinger, TINS, p. 392 (1985); and Evans et al., J.Med. Chem., 30:1229 (1987), which are incorporated herein by referencefor any purpose. Such compounds are often developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides can be used to produce asimilar therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biochemical property or pharmacological activity), such ashuman antibody, but have one or more peptide linkages optionallyreplaced by at least one linkage selected from: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH-(cis & trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) can be used in certain embodimentsto generate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation can be generated by methods known in the art (Rizo &Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein byreference for any purpose); for example, by adding internal cysteineresidues capable of forming intramolecular disulfide bridges whichcyclize the peptide.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature or a form of the materials that is found in nature.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%,about 85%, about about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or higheridentity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or through manual alignment and alsovisual inspection (see e.g., the NCBI websitehttp://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are thensaid to be “substantially identical.” This definition also refers to, ormay be applied to, the compliment of a test sequence. The definitionalso includes sequences that have deletions and/or additions, as well asthose that have substitutions. As described herein, the algorithms canaccount for gaps, and the like. In various embodiments, identity existsover a region that is at least about 25 amino acids, about 50 aminoacids or nucleotides in length, or over a region that is 50-100 aminoacids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous positions as desired. In some embodiments the“comparison window” can be selected from the group consisting of fromabout 50 to about 200, or about 100 to about 150, or greater than 150,if so desired in which a sequence may be compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of various embodiments. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences can depend upon the hostorganism. In particular embodiments, control sequences for prokaryotescan include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes caninclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” can includeleader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct can include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto. The expression vectors useful invarious embodiments described herein can contain at least one expressioncontrol sequence that is operatively linked to the DNA sequence orfragment to be expressed. The control sequence is inserted in the vectorin order to control and to regulate the expression of the cloned DNAsequence. Examples of useful expression control sequences are the lacsystem, the trp system, the tac system, the trc system, major operatorand promoter regions of phage lambda, the control region of fd coatprotein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., PhoS, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See e.g.,Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MolecularCloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methodsin Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Suchtechniques can be used to introduce one or more exogenous DNA moietiesinto suitable host cells. A transfection may be transient.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAcan recombine with that of the cell by physically integrating into achromosome of the cell, or can be maintained transiently as an episomalelement without being replicated, or can replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

An “antigen-binding protein” (“ABP”) as used herein means any proteinthat binds a specified target antigen. In this specification, thespecified target antigen is the apelin protein or fragment or regionthereof “Antigen-binding protein” includes but is not limited toantibodies and binding parts thereof, such as immunologically functionalfragments. Peptibodies are another example of antigen-binding proteins.

The term “immunologically functional fragment” (or simply “fragment”) ofan antibody or immunoglobulin chain (heavy or light chain)antigen-binding protein, as used herein, is a species of antigen-bindingprotein comprising a portion (regardless of how that portion is obtainedor synthesized) of an antibody that lacks at least some of the aminoacids present in a full-length chain but which is still capable ofspecifically binding to an antigen.

“Specific binding” should be understood to mean that the predominantantigen bound by the antigen-binding protein is, the full length apelinmolecule (SEQ ID NOs: 3-6) or a fragment thereof (e.g. SEQ ID NO: 31 or32). This does not necessarily preclude, however, binding of anantigen-binding protein to proteins other than apelin. In variousembodiments, the binding to other proteins represents less than about5%, less than about 10%, less than about 15%, less than about 20% orless than about 25% of the total protein bound.

Fragments of antigen-binding proteins are biologically active in thatthey bind to the target antigen and can compete with otherantigen-binding proteins, including intact antibodies, for binding to agiven epitope or antigen. In some embodiments, the fragments areneutralizing fragments. In some embodiments, the fragments can block orreduce the likelihood of the interaction between apelin and APJ. In oneaspect, such a fragment will retain at least one CDR present in thefull-length light or heavy chain, and in some embodiments will comprisea single heavy chain and/or light chain or portion thereof. Thesebiologically active fragments can be produced by recombinant DNAtechniques, or can be produced by enzymatic or chemical cleavage ofantigen-binding proteins, including intact antibodies. Immunologicallyfunctional immunoglobulin fragments include, but are not limited to,Fab, a diabody (heavy chain variable domain on the same polypeptide as alight chain variable domain, connected via a short peptide linker thatis too short to permit pairing between the two domains on the samechain), Fab′, F(ab′)₂, Fv, domain antibodies and single-chainantibodies, and can be derived from any mammalian source, including butnot limited to human, mouse, rat, camelid or rabbit. It is furthercontemplated that a functional portion of the antigen-binding proteinsdisclosed herein, for example, one or more CDRs, could be covalentlybound to a second protein or to a small molecule to create a therapeuticagent directed to a particular target in the body, possessingbifunctional therapeutic properties, or having a prolonged serumhalf-life. As will be appreciated by one of skill in the art, anantigen-binding protein can include nonprotein components.

Certain antigen-binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen-binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. In some embodiments, the antigen-binding proteincomprises or consists of avimers (tightly binding peptide).

An “Fc” region comprises two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

A “Fab fragment” comprises one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” comprises one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are incorporated by reference.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody cantarget the same or different antigens.

A “bivalent antigen-binding protein” or “bivalent antibody” comprisestwo antigen-binding sites. In some instances, the two binding sites havethe same antigen specificities. Bivalent antigen-binding proteins andbivalent antibodies can be bispecific as defined herein. A bivalentantibody other than a “multispecific” or “multifunctional” antibody, incertain embodiments, typically is understood to have each of its bindingsites identical.

A “multispecific antigen-binding protein” or “multispecific antibody” isone that targets more than one antigen or epitope.

A “bispecific,” “dual-specific,” or “bifunctional” antigen-bindingprotein or antibody is a hybrid antigen-binding protein or antibody,respectively, having two different antigen-binding sites. Bispecificantigen-binding proteins and antibodies are a species of multispecificantigen-binding protein antibody and can be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See e.g., Songsivilai and Lachmann, 1990, Clin. Exp.Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.The two binding sites of a bispecific antigen-binding protein orantibody will bind to two different epitopes, which can reside on thesame or different protein targets.

Each individual immunoglobulin chain is typically composed of several“immunoglobulin domains.” These domains are the basic units of whichantibody polypeptides are composed. In humans, the IgA and IgD isotypescontain four heavy chains and four light chains; the IgG and IgEisotypes contain two heavy chains and two light chains; and the IgMisotype contains five heavy chains and five light chains. The heavychain C region typically comprises one or more domains that can beresponsible for effector function. The number of heavy chain constantregion domains will depend on the isotype. IgG heavy chains, forexample, contain three C region domains known as C_(H)1, C_(H)2 andC_(H)3. The antibodies that are provided can have any of these isotypesand subtypes

“Antigen-binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen (e.g., a paratope). Forexample, that portion of an antigen-binding protein that contains theamino acid residues that interact with an antigen and confer on theantigen-binding protein its specificity and affinity for the antigen isreferred to as “antigen-binding region.” An antigen-binding regiontypically includes one or more Complementary Binding Regions (CDRs).Certain antigen-binding regions also include one or more “framework”regions. A “CDR” is an amino acid sequence that contributes toantigen-binding specificity and affinity. “Framework” regions can aid inmaintaining the proper conformation of the CDRs to promote bindingbetween the antigen-binding region and an antigen. Structurally,framework regions can be located in antibodies between CDRs.

In certain aspects, recombinant antigen-binding proteins that bindapelin, are provided. In this context, a “recombinant antigen-bindingprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as described herein.Methods and techniques for the production of recombinant proteins arewell known in the art.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An“antibody” is a species of an antigen-binding protein. An intactantibody will generally comprise at least two full-length heavy chainsand two full-length light chains, but in some instances can includefewer chains such as antibodies naturally occurring in camelids whichcan comprise only heavy chains. Antibodies can be derived solely from asingle source, or can be “chimeric,” that is, different portions of theantibody can be derived from two different antibodies as describedfurther below. The antigen-binding proteins, antibodies, or bindingfragments can be produced in hybridomas, by recombinant DNA techniques,or by enzymatic or chemical cleavage of intact antibodies. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below. Furthermore, unless explicitlyexcluded, antibodies include monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. In some embodiments, the term also encompassespeptibodies.

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”and one full-length “heavy” chain. The amino-terminal portion of eachchain typically includes a variable region that typically is responsiblefor antigen recognition. The carboxy-terminal portion of each chaintypically defines a constant region that can be responsible for effectorfunction. The variable regions of each light/heavy chain pair typicallyform the antigen-binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions orCDRs. The CDRs from the two chains of each pair typically are aligned bythe framework regions, which can enable binding to a specific epitope.From N-terminal to C-terminal, both light and heavy chain variableregions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987);Chothia et al., Nature, 342:878-883 (1989).

In certain embodiments, an antibody heavy chain binds to an antigen inthe absence of an antibody light chain. In certain embodiments, anantibody light chain binds to an antigen in the absence of an antibodyheavy chain. In certain embodiments, an antibody binding region binds toan antigen in the absence of an antibody light chain. In certainembodiments, an antibody binding region binds to an antigen in theabsence of an antibody heavy chain. In certain embodiments, anindividual variable region specifically binds to an antigen in theabsence of other variable regions.

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDR regions. Examples of such methods include, but arenot limited to, the Kabat definition, the Chothia definition, the “AbM”definition and the contact definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See e.g.,Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986);Chothia et al., Nature, 342: 877-83 (1989). The “AbM” definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See e.g., Martin et al., Proc. Natl.Acad. Sci. (USA), 86:9268-9272 (1989); “AbM™, A Computer Program forModeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular,Ltd. The AbM definition models the tertiary structure of an antibodyfrom primary sequence using a combination of knowledge databases and abinitio methods, such as those described by Samudrala et al., “Ab InitioProtein Structure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Thecontact definition is based on an analysis of the available complexcrystal structures. See e.g., MacCallum et al., J. Mol. Biol., 5:732-45(1996).

By convention, the CDR regions in the heavy chain are typically referredto as H1, H2, and H3 and are numbered sequentially in the direction fromthe amino terminus to the carboxy terminus. The CDR regions in the lightchain are typically referred to as L1, L2, and L3 and are numberedsequentially in the direction from the amino terminus to the carboxyterminus.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, V_(L), and a constant region domain, C_(L). The variable regiondomain of the light chain is at the amino-terminus of the polypeptide.Light chains include kappa chains and lambda chains.

Specificity of antibodies in various embodiments or fragments thereof,for apelin can be determined based on affinity and/or avidity. Affinity,represented by the equilibrium constant for the dissociation of anantigen with an antibody (Kd), measures the binding strength between anantigenic determinant and an antibody-binding site. Avidity is themeasure of the strength of binding between an antibody with its antigen.Avidity is related to both the affinity between an epitope with itsantigen-binding site on the antibody, and the valence of the antibody,which refers to the number of antigen-binding sites specific for aparticular epitope. The lesser the value of the Kd, the stronger thebinding strength between an antigenic determinant and the antibodybinding site.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, V_(H), and three constant region domains, C_(H)1, C_(H)2, andC_(H)3. The V_(H) domain is at the amino-terminus of the polypeptide,and the C_(H) domains are at the carboxyl-terminus, with the C_(H)3being closest to the carboxy-terminus of the polypeptide. Heavy chainscan be of any isotype, including IgG (including IgG1, IgG2, IgG3 andIgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See e.g., Songsivilai et al., Clin. Exp. Immunol.,79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

Some species of mammals can also produce antibodies having only a singleheavy chain.

Each individual immunoglobulin chain is typically composed of several“immunoglobulin domains.” These domains are the basic units of whichantibody polypeptides are composed. The heavy chain C region typicallycomprises one or more domains that can be responsible for effectorfunction. The number of heavy chain constant region domains will dependon the isotype. The antibodies that are provided can have any ofisotypes and subtypes.

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody. In certain embodiments,variable regions of different antibodies differ extensively in aminoacid sequence even among antibodies of the same species. The variableregion of an antibody typically determines specificity of a particularantibody for its target

The term “neutralizing antigen-binding protein” or “neutralizingantibody” refers to an antigen-binding protein or antibody,respectively, that binds to a ligand and prevents or reduces the bindingof the ligand to a binding partner. This can be done, for example, bydirectly blocking a binding site on the ligand or by binding to theligand and altering the ligand's ability to bind through indirect means(such as structural or energetic alterations in the ligand). In someembodiments, the term can also denote an antigen-binding protein thatprevents the protein to which it is bound from performing a biologicalfunction. In assessing the binding and/or specificity of anantigen-binding protein, e.g., an antibody or immunologically functionalfragment thereof, an antibody or fragment can substantially inhibitbinding of a ligand to its binding partner when an excess of antibodyreduces the quantity of binding partner bound to the ligand by at leastabout 1-20, about 20-30%, about 30-40%, about 40-50%, about 50-60%,about 60-70%, about 70-80%, about 80-85%, about 85-90%, about 90-95%,about 95-97%, about 97-98%, about 98-99% or more (as measured in an invitro competitive binding assay). In some embodiments, in the case ofapelin antigen-binding proteins, such a neutralizing molecule candiminish the ability of apelin to bind the APJ. In some embodiments, theneutralizing ability is characterized and/or described via a competitionassay. In some embodiments, the neutralizing ability is described interms of an IC₅₀ or EC₅₀ value. In some embodiments, the antigen-bindingproteins may be non-neutralizing antigen-binding proteins.

The term “target” refers to a molecule or a portion of a moleculecapable of being bound by an antigen-binding protein. In certainembodiments, a target can have one or more epitopes. In certainembodiments, a target is an antigen. The use of “antigen” in the phrase“antigen-binding protein” simply denotes that the protein sequence thatcomprises the antigen can be bound by an antibody. In this context, itdoes not require that the protein be foreign or that it be capable ofinducing an immune response.

The term “compete” when used in the context of antigen-binding proteins(e.g., neutralizing antigen-binding proteins or neutralizing antibodies)that compete for the same epitope means competition betweenantigen-binding proteins as determined by an assay in which theantigen-binding protein (e.g., antibody or immunologically functionalfragment thereof) being tested prevents or inhibits (e.g., reduces)specific binding of a reference antigen-binding protein (e.g., a ligand,or a reference antibody) to a common antigen (e.g., apelin or a fragmentthereof). Numerous types of competitive binding assays can be used todetermine if one antigen-binding protein competes with another, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253);solid phase direct biotin-avidin EIA (see e.g., Kirkland et al., 1986,J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see e.g., Harlow and Lane, 1988,Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using 1-125 label (see e.g., Morel et al., 1988, Molec.Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see e.g.,Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA(Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabelled test antigen-bindingprotein and a labeled reference antigen-binding protein. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test antigen-bindingprotein. Usually the test antigen-binding protein is present in excess.Antigen-binding proteins identified by competition assay (competingantigen-binding proteins) include antigen-binding proteins binding tothe same epitope as the reference antigen-binding proteins andantigen-binding proteins binding to an adjacent epitope sufficientlyproximal to the epitope bound by the reference antigen-binding proteinfor steric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the examples herein.Usually, when a competing antigen-binding protein is present in excess,it will inhibit (e.g., reduce) specific binding of a referenceantigen-binding protein to a common antigen by at least about 40-45%,about 45-50%, about about 50-55%, about 55-60%, about 60-65%, about65-70%, about 70-75% or about 75% or more. In some instances, binding isinhibited by at least about 80-85%, about 85-90%, about 90-95%, about95-97%, or about 97% or more.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantigen-binding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof). In some embodiments, the antigen iscapable of being used in an animal to produce antibodies capable ofbinding to that antigen. An antigen can possess one or more epitopesthat are capable of interacting with different antigen-binding proteins,e.g., antibodies.

The term “epitope” includes any determinant capable being bound by anantigen-binding protein, such as an antibody or to a T-cell receptor. Anepitope is a region of an antigen that is bound by an antigen-bindingprotein that targets that antigen, and when the antigen is a protein,includes specific amino acids that directly contact the antigen-bindingprotein. Most often, epitopes reside on proteins, but in some instancescan reside on other kinds of molecules, such as nucleic acids. Epitopedeterminants can include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl or sulfonylgroups, and can have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. Generally,antibodies specific for a particular target antigen will preferentiallyrecognize an epitope on the target antigen in a complex mixture ofproteins and/or macromolecules.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least about 50% (ona molar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at leastabout 80%, about 85%, about 90%, about 95%, or about 99% of allmacromolecular species present in the composition. In other embodiments,the object species is purified to essential homogeneity whereincontaminating species cannot be detected in the composition byconventional detection methods and thus the composition consists of asingle detectable macromolecular species.

The term “biological sample”, as used herein, includes, but is notlimited to, any quantity of a substance from a living thing or formerlyliving thing. Such living things include, but are not limited to,humans, mice, monkeys, rats, rabbits, and other animals. Such substancesinclude, but are not limited to, blood, serum, urine, cells, organs,tissues, bone, bone marrow, lymph nodes, and skin.

The term “pharmaceutical agent composition” (or agent or drug) as usedherein refers to a chemical compound, composition, agent or drug capableof inducing a desired therapeutic effect when properly administered to apatient. It does not necessarily require more than one type ofingredient.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose” refer to the amount of an apelin antigen-binding proteindetermined to produce a therapeutic response in a mammal Suchtherapeutically effective amounts can be ascertained by one of ordinaryskill in the art. The exact dose and formulation will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Remington: The Science andPractice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar,Dosage Calculations (1999)).

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.

The term “modulator,” as used herein, is a compound that changes oralters the activity or function of a molecule. For example, a modulatorcan cause an increase or decrease in the magnitude of a certain activityor function of a molecule compared to the magnitude of the activity orfunction observed in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Certain exemplaryactivities and functions of a molecule include, but are not limited to,binding affinity, enzymatic activity, and signal transduction. Certainexemplary inhibitors include, but are not limited to, proteins,peptides, antigen-binding fragments, antibodies, peptibodies,carbohydrates or small organic molecules. An antibody can be madeagainst apelin. Peptibodies are described in, e.g., U.S. Pat. No.6,660,843 (corresponding to PCT Application No. WO 01/83525).

The terms “patient” and “subject” are used interchangeably and includehuman and non-human animal subjects as well as those with formallydiagnosed disorders, those without formally recognized disorders, thosereceiving medical attention, those at risk of developing the disorders,etc.

The term “treat” and “treatment” includes therapeutic treatments,prophylactic treatments, and applications in which one reduces the riskthat a subject will develop a disorder or other risk factor. Treatmentdoes not require the complete curing of a disorder and encompassesembodiments in which one reduces symptoms or underlying risk factors.

The term “prevent” does not require the 100% elimination of thepossibility of an event. Rather, it denotes that the likelihood of theoccurrence of the event has been reduced in the presence of the compoundor method.

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques can beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

Antigen-binding proteins (ABPs) that bind apelin, are provided herein.In some embodiments, the antigen-binding proteins provided arepolypeptides which comprise one or more complementary determiningregions (CDRs), as described herein. In some antigen-binding proteins,the CDRs are embedded into a “framework” region, which orients theCDR(s) such that the proper antigen-binding properties of the CDR(s) isachieved. In some embodiments, antigen-binding proteins provided hereincan interfere with, block, reduce or modulate the interaction betweenapelin and APJ. Such antigen-binding proteins are denoted as“neutralizing.” In some embodiments, the neutralizing antigen-bindingprotein binds to apelin in a location and/or manner that prevents apelinfrom binding to APJ.

In some embodiments, the antigen-binding proteins provided herein arecapable of inhibiting apelin-mediated activity (including binding). Insome embodiments, antigen-binding proteins binding to an apelin epitopecan inhibit, inter alia, interactions between apelin and APJ and otherphysiological effects mediated by the apelin/APJ interaction. In someembodiments, the antigen-binding proteins are chimeras, such as ahuman/mouse chimera.

In some embodiments, the antigen-binding protein can bind to the matureform of apelin. In other embodiments the antigen-binding protein canbind in the prodomain of apelin.

The antigen-binding proteins that are disclosed herein have a variety ofutilities. Some of the antigen-binding proteins, for instance, areuseful in specific binding assays or for affinity purification ofapelin, Some of the antigen-binding proteins may be useful forinhibiting binding of apelin to APJ, or inhibiting apelin/APJ-mediatedactivities. In some embodiments, binding of and antigen-binding proteinto apelin can prevent angiogenesis. If angiogenesis is prevented thismay treat retinopathy or cancer.

The antigen-binding proteins can be used in a variety of therapeuticapplications, as explained herein. For example, in some embodiments theapelin antigen-binding proteins are useful for treating diseases andconditions associated with apelin and/or APJ such as diabeticretinopathy, cancer, or other pathologic disorder associated withangiogenesis.

In some embodiments, the antigen-binding proteins that are providedcomprise one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs). In someembodiments, the antigen-binding protein comprises (a) a polypeptidestructure and (b) one or more CDRs that are inserted into and/or joinedto the polypeptide structure. The polypeptide structure can take avariety of different forms. For example, it can be, or comprise, theframework of a naturally occurring antibody, or fragment or variantthereof, or can be completely synthetic in nature.

In certain embodiments, the polypeptide structure of the antigen-bindingproteins is an antibody or is derived from an antibody, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the antigen-binding protein is an immunological fragment ofan antibody (e.g., a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment,an Fv fragment, a diabody, or a single chain antibody molecule, such asan scFv)

In embodiments where the antigen-binding protein is used for therapeuticapplications, an antigen-binding protein can inhibit, interfere with ormodulate one or more biological activities of apelin. In one embodiment,an antigen-binding protein binds specifically to human apelin and/orsubstantially inhibits binding of human apelin to APJ by at least about20%-40%, about 40-60%, about 60-80%, about 80-85%, or more (for example,by measuring binding in an in vitro competitive binding assay).

Some of the antigen-binding proteins that are provided herein areantibodies. In some embodiments, the antigen-binding protein has a K_(d)of less (binding more tightly) than about 10⁻⁷, about 10⁻⁸, about 10⁻⁹,about 10⁻¹⁰, about 10⁻¹¹, about 10⁻¹², about 10⁻¹³M. In someembodiments, the antigen-binding protein has an IC₅₀ for blocking thebinding of apelin to APJ of less than about 1 μM, about 1000 nM to about100 nM, about 100 nM to about 10 nM, about about 10 nM to about 1 nM,about 1000 pM to about 500 pM, about 500 pM to about 200 pM, less thanabout 200 pM, about 200 pM to about 150 pM, about 200 pM to about 100pM, about 100 pM to about 10 pM, about 10 pM to about 1 pM.

In some embodiments, the antigen-binding proteins bind to a specificconformational state of apelin to prevent apelin from interacting withAPJ. When apelin is prevented from interacting with APJ, this canprevent or block apelin or APJ mediated activity and the resultantpathology resulting from the apelin/APJ interaction.

As described herein, an antigen-binding protein to apelin can comprise ahumanized antibody and/or part thereof. A practical application of sucha strategy is the “humanization” of the mouse humoral immune system.

In certain embodiments, a humanized antibody is substantiallynon-immunogenic in humans. In certain embodiments, a humanized antibodyhas substantially the same affinity for a target as an antibody fromanother species from which the humanized antibody is derived. See e.g.,U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; and U.S. Pat. No. 5,585,089.

In certain embodiments, amino acids of an antibody variable domain thatcan be modified without diminishing the native affinity of theantigen-binding domain while reducing its immunogenicity are identified.See e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619.

In certain embodiments, modification of an antibody by methods known inthe art is typically designed to achieve increased binding affinity fora target and/or to reduce immunogenicity of the antibody in therecipient. In certain embodiments, humanized antibodies can be modifiedto eliminate glycosylation sites in order to increase affinity of theantibody for its cognate antigen. See e.g., Co et al., Mol. Immunol.,30:1361-1367 (1993). In certain embodiments, techniques such as“reshaping,” “hyperchimerization,” or “veneering/resurfacing” are usedto produce humanized antibodies. See e.g., Vaswami et al., Annals ofAllergy, Asthma, & Immunol. 81:105 (1998); Roguska et al., Prot. Engin.,9:895-904 (1996); and U.S. Pat. No. 6,072,035. In certain suchembodiments, such techniques typically reduce antibody immunogenicity byreducing the number of foreign residues, but do not preventanti-idiotypic and anti-allotypic responses following repeatedadministration of the antibodies. Certain other methods for reducingimmunogenicity are described, e.g., in Gilliland et al., J. Immunol.,62(6):3663-71 (1999).

In certain instances, humanizing antibodies can result in a loss ofantigen-binding capacity. The humanized antibodies can then be “backmutated.” In such embodiments, the humanized antibody can be mutated toinclude one or more of the amino acid residues found in the donorantibody. See e.g., Saldanha et al., Mol. Immunol. 36:709-19 (1999).

In certain embodiments the complementarity determining regions (CDRs) ofthe light and heavy chain variable regions of an antibody to apelin canbe grafted to framework regions (FRs) from the same, or another,species. In certain embodiments, the CDRs of the light and heavy chainvariable regions of an antibody to apelin can be grafted to consensushuman FRs. To create consensus human FRs, in certain embodiments, FRsfrom several human heavy chain or light chain amino acid sequences arealigned to identify a consensus amino acid sequence. In certainembodiments, the FRs of an antibody to apelin heavy chain or light chainare replaced with the FRs from a different heavy chain or light chain.In certain embodiments, rare amino acids in the FRs of the heavy andlight chains of an antibody to apelin are not replaced, while the restof the FR amino acids are replaced. Rare amino acids are specific aminoacids that are in positions in which they are not usually found in FRs.In certain embodiments, the grafted variable regions from an antibody toapelin can be used with a constant region that is different from theconstant region of an antibody to apelin. In certain embodiments, thegrafted variable regions are part of a single chain Fv antibody. CDRgrafting is described, e.g., in U.S. Pat. Nos. 6,180,370, 6,054,297,5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101,and in Jones et al., Nature, 321: 522-525 (1986); Riechmann et al.,Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988), Winter, FEBS Letts., 430:92-94 (1998), which are herebyincorporated by reference for any purpose.

In certain embodiments, antigen-binding proteins (such as antibodies)are produced by immunization with an antigen (e.g., apelin or a fragmentthereof). The antibodies can be produced by immunization withfull-length apelin, a soluble form of apelin, the catalytic domainalone, the mature form of apelin, a splice variant form of apelin, or afragment thereof. In certain embodiments, the antibodies of can bepolyclonal or monoclonal, and/or can be recombinant antibodies

In certain embodiments, strategies can be employed to manipulateinherent properties of an antibody, such as the affinity of an antibodyfor its target. Such strategies include, but are not limited to, the useof site-specific or random mutagenesis of the polynucleotide moleculeencoding an antibody to generate an antibody variant. In certainembodiments, such generation is followed by screening for antibodyvariants that exhibit the desired change, e.g. increased or decreasedaffinity.

In certain embodiments, the amino acid residues targeted in mutagenicstrategies are those in the CDRs. In other embodiments, amino acids inthe framework regions of the variable domains can be targeted. Suchframework regions have been shown to contribute to the target bindingproperties of certain antibodies. See e.g., Hudson, Curr. Opin.Biotech., 9:395-402 (1999) and references therein.

In certain embodiments, smaller and more effectively screened librariesof antibody variants can be produced by restricting random orsite-directed mutagenesis to hyper-mutation sites in the CDRs, which aresites that correspond to areas prone to mutation during the somaticaffinity maturation process. See e.g., Chowdhury & Pastan, NatureBiotech., 17: 568-572 (1999) and references therein. In certainembodiments, certain types of DNA elements can be used to identifyhyper-mutation sites including, but not limited to, certain direct andinverted repeats, certain consensus sequences, certain secondarystructures, and certain palindromes. For example, such DNA elements thatcan be used to identify hyper-mutation sites include, but are notlimited to, a tetrabase sequence comprising a purine (A or G), followedby guanine (G), followed by a pyrimidine (C or T), followed by eitheradenosine or thymidine (A or T) (i.e., A/G-G-C/T-A/T). Another exampleof a DNA element that can be used to identify hyper-mutation sites isthe serine codon, A-G-C/T.

For preparation of suitable antibodies for various embodiments e.g.,recombinant, monoclonal, or polyclonal antibodies, many techniques knownin the art can be used (see e.g., Kohler & Milstein, Nature 256:495-497(1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane,Antibodies, A Laboratory Manual (1988); and Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986)). The genes encodingthe heavy and light chains of an antibody of interest can be cloned froma cell, e.g., the genes encoding a monoclonal antibody can be clonedfrom a hybridoma and used to produce a recombinant monoclonal antibody.Gene libraries encoding heavy and light chains of monoclonal antibodiescan also be made from hybridoma or plasma cells. Random combinations ofthe heavy and light chain gene products generate a large pool ofantibodies with different antigenic specificity (see e.g., Kuby,Immunol. (3^(rd) ed. 1997)). Techniques for the production of singlechain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778,U.S. Pat. No. 4,816,567) can be adapted to produce antibodies topolypeptides for various embodiments. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanized orhuman antibodies (see e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al.,Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859(1994); Morrison, Nature, 368:812-13 (1994); Fishwild et al., NatureBiotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826(1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies canalso be made bispecific, i.e., able to recognize two different antigens(see e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991);and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies canalso be heteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see e.g., Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (e.g. a gamma constant region) areformed into a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg & Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort &Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns etal., and U.S. Pat. No. 5,643,763 to Choi & Dunn, and GenPharmInternational U.S. patent application Ser. Nos. 07/574,748, 07/575,962,07/810,279, 07/853,408, 07/904,068, 07/990,860, 08/053,131, 08/096,762,08/155,301, 08/161,739, 08/165,699, 08/209,741, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety.

The antibodies can be fused to additional amino acid residues. Suchamino acid residues can be a peptide tag, perhaps to facilitateisolation. Other amino acid residues for homing of the antibodies tospecific organs or tissues are also contemplated.

In certain embodiments the antibody or the antigen-binding region of anyof the monoclonal antibodies described herein can be used to treatcancer or retinopathy.

“Cancer” should be understood to be a general term that can be used toindicate any of various types of malignant neoplasms, which may invadesurrounding tissues, may metastasize to several sites and may likelyrecur after attempted removal. The term may also refer to any carcinomaor sarcoma.

“Retinopathy” should be understood to mean a non-inflammatory disease ofthe retina, as distinguished from retinitis. “Diabetic retinopathy”should be understood to mean retinal changes occurring in diabetes, thatcan be marked by punctuate hemorrhages, microaneurysms and sharplydefined waxy exudates.

In treating cancer, the antigen-binding region can be joined to at leasta functionally active portion of a second protein having therapeuticactivity. The second protein can include, but is not limited to, anenzyme, lymphokine, oncostatin or toxin. Suitable toxins includedoxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin(PE) A, PE40, ricin, abrin, glucocorticoid and radioisotopes.

As will be appreciated, antibodies can be expressed in cell lines otherthan hybridoma cell lines. Sequences encoding particular antibodies canbe used to transform a suitable mammalian host cell. Transformation canbe by any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroducing heterologous polynucleotides into mammalian cells are wellknown in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), human epithelial kidney 293 cells, and a number of othercell lines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels of the antibodyof interest.

In certain embodiments, antigen-binding proteins can comprise animmunoglobulin molecule of at least one of the IgG1, IgG2, IgG3, IgG4,Ig E, IgA, IgD, and IgM isotype. In certain embodiments, antigen-bindingproteins comprise a human kappa light chain and/or a human heavy chain.In certain embodiments, the heavy chain is of the IgG1, IgG2, IgG3,IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments,antigen-binding proteins have been cloned for expression in mammaliancells. In certain embodiments, antigen-binding proteins comprise aconstant region other than any of the constant regions of the IgG1,IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype.

In certain embodiments, substantial modifications in the functionaland/or chemical characteristics of antibodies to apelin can beaccomplished by selecting substitutions in the amino acid sequence ofthe heavy and light chains that differ significantly in their effect onmaintaining (a) the structure of the molecular backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain.

For example, a “conservative amino acid substitution” can involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide can also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of antibodiesto apelin, or to increase or decrease the affinity of the antibodies toapelin as described herein.

In certain embodiments, antibodies or antigen-binding proteins can beexpressed in cell lines other than hybridoma cell lines. Sequencesencoding particular antibodies can be used for transformation of asuitable mammalian host cell. According to certain embodiments,transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference for any purpose). In certain embodiments, thetransformation procedure used can depend upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are well known in the art and include, but are notlimited to, dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines can be selected throughdetermining which cell lines have high expression levels and produceantibodies with constitutive HGF binding properties. Appropriateexpression vectors for mammalian host cells are well known.

In certain embodiments, antigen-binding proteins comprise one or morepolypeptides. Any of a variety of expression vector/host systems can beutilized to express polynucleotide molecules encoding polypeptidescomprising one or more antigen-binding protein components or theantigen-binding protein itself. Such systems include, but are notlimited to, microorganisms, such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or animalcell systems.

In certain embodiments, a polypeptide comprising one or moreantigen-binding protein components or the antigen-binding protein itselfis recombinantly expressed in yeast. Certain such embodiments usecommercially available expression systems, e.g., the Pichia ExpressionSystem (Invitrogen, San Diego, Calif.), following the manufacturer'sinstructions. In certain embodiments, such a system relies on thepre-pro-alpha sequence to direct secretion. In certain embodiments,transcription of the insert is driven by the alcohol oxidase (AOX1)promoter upon induction by methanol.

In certain embodiments, a secreted polypeptide comprising one or moreantigen-binding protein components or the antigen-binding protein itselfis purified from yeast growth medium. In certain embodiments, themethods used to purify a polypeptide from yeast growth medium is thesame as those used to purify the polypeptide from bacterial andmammalian cell supernatants.

In certain embodiments, a nucleic acid encoding a polypeptide comprisingone or more antigen-binding protein components or the antigen-bindingprotein itself is cloned into a baculovirus expression vector, such aspVL1393 (PharMingen, San Diego, Calif.). In certain embodiments, such avector can be used according to the manufacturer's directions(PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-freemedia and to produce recombinant polypeptide. In certain embodiments, apolypeptide is purified and concentrated from such media using aheparin-Sepharose column (Pharmacia).

In certain embodiments, a polypeptide comprising one or moreantigen-binding protein components or the antigen-binding protein itselfis expressed in an insect system. Certain insect systems for polypeptideexpression are well known to those of skill in the art. In one suchsystem, Autographa californica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes in Spodoptera frugiperda cellsor in Trichoplusia larvae. In certain embodiments, a nucleic acidmolecule encoding a polypeptide can be inserted into a nonessential geneof the virus, for example, within the polyhedrin gene, and placed undercontrol of the promoter for that gene. In certain embodiments,successful insertion of a nucleic acid molecule will render thenonessential gene inactive. In certain embodiments, that inactivationresults in a detectable characteristic. For example, inactivation of thepolyhedrin gene results in the production of virus lacking coat protein.

In certain embodiments, recombinant viruses can be used to infect S.frugiperda cells or Trichoplusia larvae. See e.g., Smith et al., J.Virol., 46: 584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA),91: 3224-7 (1994).

In certain embodiments, polypeptides comprising one or moreantigen-binding protein components or the antigen-binding protein itselfmade in bacterial cells are produced as insoluble inclusion bodies inthe bacteria. Host cells comprising such inclusion bodies are collectedby centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA;and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15minutes at room temperature. In certain embodiments, the lysate iscleared by sonication, and cell debris is pelleted by centrifugation for10 minutes at 12,000×g. In certain embodiments, thepolypeptide-containing pellet is resuspended in 50 mM Tris, pH 8, and 10mM EDTA; layered over 50% glycerol; and centrifuged for 30 minutes at6000×g. In certain embodiments, that pellet can be resuspended instandard phosphate buffered saline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺.In certain embodiments, the polypeptide is further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (see e.g., Sambrook et al., supra). In certain embodiments, such agel can be soaked in 0.4 M KCl to visualize the protein, which can beexcised and electroeluted in gel-running buffer lacking SDS. Accordingto certain embodiments, a Glutathione-S-Transferase (GST) fusion proteinis produced in bacteria as a soluble protein. In certain embodiments,such GST fusion protein is purified using a GST Purification Module(Pharmacia).

In certain embodiments, it is desirable to “refold” certainpolypeptides, e.g., polypeptides comprising one or more antigen-bindingprotein components or the antigen-binding protein itself. In certainembodiments, such polypeptides are produced using certain recombinantsystems discussed herein. In certain embodiments, polypeptides are“refolded” and/or oxidized to form desired tertiary structure and/or togenerate disulfide linkages. In certain embodiments, such structureand/or linkages are related to certain biological activity of apolypeptide. In certain embodiments, refolding is accomplished using anyof a number of procedures known in the art. Exemplary methods include,but are not limited to, exposing the solubilized polypeptide agent to apH typically above 7 in the presence of a chaotropic agent. An exemplarychaotropic agent is guanidine. In certain embodiments, therefolding/oxidation solution also contains a reducing agent and theoxidized form of that reducing agent. In certain embodiments, thereducing agent and its oxidized form are present in a ratio that willgenerate a particular redox potential that allows disulfide shuffling tooccur. In certain embodiments, such shuffling allows the formation ofcysteine bridges. Exemplary redox couples include, but are not limitedto, cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride,dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME.In certain embodiments, a co-solvent is used to increase the efficiencyof refolding. Exemplary cosolvents include, but are not limited to,glycerol, polyethylene glycol of various molecular weights, andarginine.

In certain embodiments, one substantially purifies a polypeptidecomprising one or more antigen-binding protein components or theantigen-binding protein itself. Certain protein purification techniquesare known to those of skill in the art. In certain embodiments, proteinpurification involves crude fractionation of polypeptide fractionationsfrom non-polypeptide fractions. In certain embodiments, polypeptides arepurified using chromatographic and/or electrophoretic techniques.Exemplary purification methods include, but are not limited to,precipitation with ammonium sulphate; precipitation with PEG;immunoprecipitation; heat denaturation followed by centrifugation;chromatography, including, but not limited to, affinity chromatography(e.g., Protein-A-Sepharose), ion exchange chromatography, exclusionchromatography, and reverse phase chromatography; gel filtration;hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gelelectrophoresis; and combinations of such and other techniques. Incertain embodiments, a polypeptide is purified by fast protein liquidchromatography or by high pressure liquid chromatography (HPLC). Incertain embodiments, purification steps can be changed or certain stepscan be omitted, and still result in a suitable method for thepreparation of a substantially purified polypeptide.

In certain embodiments, one quantitates the degree of purification of apolypeptide preparation. Certain methods for quantifying the degree ofpurification are known to those of skill in the art. Certain exemplarymethods include, but are not limited to, determining the specificbinding activity of the preparation and assessing the amount of apolypeptide within a preparation by SDS/PAGE analysis. Certain exemplarymethods for assessing the amount of purification of a polypeptidepreparation comprise calculating the binding activity of a preparationand comparing it to the binding activity of an initial extract. Incertain embodiments, the results of such a calculation are expressed as“fold purification.” The units used to represent the amount of bindingactivity depend upon the particular assay performed.

In certain embodiments, a polypeptide comprising one or moreantigen-binding protein components or the antigen-binding protein itselfis partially purified. Partial purification can be accomplished by usingfewer purification steps or by utilizing different forms of the samegeneral purification scheme. For example, in certain embodiments,cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “fold purification” thanthe same technique utilizing a low-pressure chromatography system. Incertain embodiments, methods resulting in a lower degree of purificationcan have advantages in total recovery of polypeptide, or in maintainingbinding activity of a polypeptide.

In certain instances, the electrophoretic migration of a polypeptide canvary, sometimes significantly, with different conditions of SDS/PAGE.See e.g., Capaldi et al., Biochem. Biophys. Res. Comm., 76: 425 (1977).It will be appreciated that under different electrophoresis conditions,the apparent molecular weights of purified or partially purifiedpolypeptide can be different.

In various embodiments described herein, antibodies can be used in vivoand in vitro for investigative or diagnostic methods, which are wellknown in the art. The diagnostic methods include kits, which containantibodies in various embodiments. In other embodiments the antibodiesdescribed herein can be used as a therapeutic.

It is understood that the anti-apelin antibodies, where used in a mammalfor the purpose of prophylaxis or treatment, can be administered in theform of a composition that additionally can comprise a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriersinclude, for example, one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof.

Pharmaceutically acceptable carriers can further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antigen-binding proteins. The compositions of the injection can,as is well known in the art, be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the mammal.

Pharmaceutical formulations, particularly, of the antibodies for usedescribed herein can be prepared by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers. Such formulations can belyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations used. Acceptable carriers, excipients or stabilizers canbe acetate, phosphate, citrate, and other organic acids; antioxidants(e.g., ascorbic acid) preservatives low molecular weight polypeptides;proteins, such as serum albumin or gelatin, or hydrophilic polymers suchas polyvinylpyllolidone; and amino acids, monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents; and ionic and non-ionic surfactants (e.g.,polysorbate); salt-forming counter-ions such as sodium; metal complexes(e.g., Zn-protein complexes); and/or non-ionic surfactants. The antibodycan be formulated at a concentration of between 0.5-200 mg/ml.

In therapeutic applications, compositions are administered to a patientsuffering from a disease (e.g., diabetic retinopathy) in a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. A “patient” or “subject” as referred toherein can include both humans and other animals, particularly mammals.Thus the methods are applicable to both human therapy and veterinaryapplications. In various embodiments the patient is a mammal. The mammalcan be a primate, or even a human.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desiredantigen-binding protein to apelin, with or without additionaltherapeutic agents, in a pharmaceutically acceptable vehicle. In certainembodiments, a vehicle for parenteral injection is sterile distilledwater in which an antigen-binding protein to apelin, with or without atleast one additional therapeutic agent, is formulated as a sterile,isotonic solution, properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, methods are provided for inhibiting angiogenesisor tumorigenesis and are used to treat a patient with disease orcondition that involves angiogenesis or tumorigenesis. As used herein,the phrase “disease or condition involving angiogenesis” is intended toinclude, but is not limited to, hemangioma, solid tumors, leukemias,lymphomas, myelomas, plaque neovascularization, corneal diseases,rubeosis, neovascular glaucoma, retinopathy, exudative age-relatedmacular degeneration (AMD), proliferative diabetic retinopathy (PDR),diabetic macular edema (DME), neovascular glaucoma, cornealneovascularization (trachoma), and pterygium, diabetic retinopathy,retrolental fibroplasia, diabetic neovascularization, maculardegeneration, uterine bleeding, endometrial hyperplasia and carcinoma,endometriosis, myometrial fibroids (uterine leiomyomas) and adenomyosis,ovarian hyperstimulation syndrome, tumorigenesis or cancer.

Other aspects of the invention will be appreciated by one skilled in theart, and are described herein. Although various embodiments of theinvention have been described herein, including the following examples,those skilled in the art will readily appreciate that the specificexamples and studies detailed herein are only illustrative. It should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

EXAMPLES Example 1

Antibody Generation

Mice were immunized with an apelin peptide (QRPRLSHKGPMPF) (SEQ ID NO:31) conjugated with KLH and then boosted for 10 times. Hybridomas werethen prepared from which monoclonal antibodies could be obtained.

Hybridomas were screened using an ELISA-based assay to identify positivebinders. The monoclonal antibody, antibody A (Ab A), was selected andpurified for further characterization of affinity binding andneutralizing activity.

The amino acid sequence of the antibody is shown in FIG. 1. The figurepresents the sequence of the light chain and heavy chain of Ab A toapelin. The CDR regions in the heavy and light chain are shown in bold,italic type. The signal sequences are underlined. Additionally, theindividual CDR's are presented.

Example 2

Epitope Mapping

The antibody binding site was mapped using an ELISA-based assay.Briefly, various biotinylated apelin peptides (SEQ ID NOs: 7-22) werecaptured onto neutravidin coated plates and incubated with apelinantibody. Bound antibody was detected with horseradishperoxidase-conjugated goat anti-mouse Fc in the presence of thechromogenic substrate. The absorbance was read at 650 nm. The Ab A wasdetermined to bind to the epitope comprising amino acids PRLSHKG (SEQ IDNO: 32) of apelin as shown in FIG. 2. Ab A is believed to neutralize allactive forms of apelin.

Example 3

Binding Affinity of Ab A

Binding affinity of Ab A was determined using BIAcore solutionequilibrium binding analysis. The results are shown in Table 2 and FIG.3. Briefly, biotin-labeled apelin was immobilized at high density on aneutravidin surface. Ab A was incubated with apelin 12 at RT for over 4hours before injected over the immobilized surfaces. The dissociationequilibrium constant (K_(d)) was obtained from a nonlinear regressionanalysis of the competition curve using a one-site homogeneous bindingmodel (KinExATM Pro software). The K_(d) value was estimated to be 6 nM.

TABLE 2 95% confidence Antibody K_(D) (nM) interval (nM) Ab A 6 4-7

Example 4

Neutralization Activity of Ab A

CHOK1 cells overexpressing the human apelin receptor/APJ were used tomeasure apelin-induced changes in cAMP levels. Cells were plated thenight before the assay at a density of 2×10⁴ cells/well in a 96-wellassay plate. Serial dilutions of Ab A (10-6 M-10-9 M) were incubatedwith 6 nM of apelin (Phoenix Pharmaceuticals, Inc.) for 20 min beforebeing added to 500 nM of forskolin and the cells. The cAMP level wasmeasured using HTRF assay kit according to the manufacture's protocol(Cisbio). The luminescence was measured using RUBY star plate reader(BMG LABTECH). The neutralizing activity of Ab A (IC₅₀) was calculatedfrom the value of peak luminescence. The result indicated that Ab Adose-dependently inhibited apelin-mediated changes in cAMP levels, withIC₅₀ about 18 nM.

Example 5

Anti-Angiogenesis Activity of Ab A

Angiogenesis activity was measured using Cellplayer GFP AngioKit-96(Essen Bioscience). Briefly, human endothelial cells were co-culturedwith other human cells in a specially designed medium and treated withapelin with or without Ab A for three days. The endothelial cellsproliferated and migrated to form threadlike tubule structures. The tubeformation, which is a morphogenic indicator of angiogenesis was measuredusing IncuCyte angiogenesis module (Essen Bioscience). Apelin stimulatedthe tube formation and Ab A inhibited the apelin-induced tube formationas clearly indicated by decreased tube formation shown in FIG. 4B.

Example 6

Treatment of Diabetic Retinopathy

A subject in need of treatment for diabetic retinopathy will be injectedintravitreal with an antibody prepared against apelin that is known toaffect angiogenesis, e.g. Ab A. The treatment will continue until thereis an improvement in the condition as indicated by a decrease inneovascularization. Such an improvement can also be determined bymeasurement of either biochemical or physiological parameters deemedappropriate by one of skill in the art. For example, see Tao et al.,Invest Opthamol Visual Sci., 51:4237-4242 (2010).

Example 7

Systemic Administration of Apelin Ab on Retinal Neovascularization

OIR (oxygen-induced retinopathy) animal model was used to examine theeffects of apelin Ab on retinal neovascularization. The IgG isotypecontrol (200 ug) or apelin Ab (200 ug, 2 ug, 0.2 ug and 0.05 ug) wasadministrated s.c. daily for 9 days starting on P8 (8 days post-natal).On P17, mice were sacrificed and eyes were enucleated. Ten sections ofeach eye from each group were stained with hematoxylin-eosin (HE) andthe nuclei extending beyond the internal limiting membrane were counted.Apelin Ab (200 ug) significantly inhibited the retinalneovascularization. (FIG. 6)

Example 8

Intravitreal Injection of Apelin Ab on Retinal Neovascularization

Following the induction of OIR by oxygen cycling, eyes of P12 mice weretreated with an intravitreal injection of saline, IgG control or apelinAb (5 and 25 ng). On P17, retinal flatmounts were stained withfluorescent-labeled isolectin B4 and retinal vascular growth wasassessed. Apelin Ab significantly reduced the neovascular area inretina. (FIG. 7)

Throughout this specification various publications, patents and patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference into thisapplication. The reference to such documents, however, should not beconstrued as an acknowledgment that such documents are prior art to theapplication. Further, merely because a document may be incorporated byreference, this does not necessarily indicate that the applicants are incomplete agreement with the document's contents.

What is claimed:
 1. A method for inhibiting retinal neovascularizationin a patient in need thereof comprising administering to the patient atherapeutically effective amount of a monoclonal antibody or fragmentthereof, wherein the monoclonal antibody or fragment thereofspecifically binds to human apelin at an epitope with the sequence ofSEQ ID NO: 32 and inhibits the binding of human apelin to the APJreceptor.
 2. The method of claim 1, wherein the monoclonal antibody orfragment thereof comprises light chain complementarity determiningregions L1, L2, and L3 and heavy chain complementarity determiningregions H1, H2, and H3, wherein L1, L2, and L3 have the amino acidsequence of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively, and wherein H1, H2, and H3 have the amino acid sequence ofSEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.
 3. Themethod of claim 1, wherein the monoclonal antibody or fragment thereofcomprises a light chain comprising the sequence of SEQ ID NO: 2 and aheavy chain comprising the sequence of SEQ ID NO:
 27. 4. The method ofclaim 1, wherein the monoclonal antibody is a humanized antibody, achimeric antibody, or a multispecific antibody.
 5. The method of claim1, wherein the patient has retinopathy.
 6. The method of claim 5,wherein the retinopathy is diabetic retinopathy.
 7. The method of claim1, wherein the patient has exudative age-related macular degeneration.8. The method of claim 1, wherein the monoclonal antibody or fragmentthereof is administered to the patient parenterally.
 9. The method ofclaim 1, wherein the monoclonal antibody or fragment thereof isadministered to the patient subcutaneously.
 10. The method of claim 1,wherein the monoclonal antibody or fragment thereof is administered tothe patient intravitreally.
 11. A method for treating retinopathy in apatient in need thereof comprising administering to the patient atherapeutically effective amount of a monoclonal antibody or fragmentthereof, wherein the monoclonal antibody or fragment thereofspecifically binds to human apelin at an epitope with the sequence ofSEQ ID NO: 32 and inhibits the binding of human apelin to the APJreceptor.
 12. The method of claim 11, wherein the monoclonal antibody orfragment thereof comprises light chain complementarity determiningregions L1, L2, and L3 and heavy chain complementarity determiningregions H1, H2, and H3, wherein L1, L2, and L3 have the amino acidsequence of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively, and wherein H1, H2, and H3 have the amino acid sequence ofSEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.
 13. Themethod of claim 11, wherein the monoclonal antibody or fragment thereofcomprises a light chain comprising the sequence of SEQ ID NO: 2 and aheavy chain comprising the sequence of SEQ ID NO:
 27. 14. The method ofclaim 11, wherein the monoclonal antibody is a humanized antibody, achimeric antibody, or a multispecific antibody.
 15. The method of claim11, wherein the monoclonal antibody or fragment thereof is administeredto the patient parenterally.
 16. The method of claim 11, wherein themonoclonal antibody or fragment thereof is administered to the patientsubcutaneously.
 17. The method of claim 11, wherein the monoclonalantibody or fragment thereof is administered to the patientintravitreally.
 18. The method of claim 11, wherein the retinopathy isdiabetic retinopathy.
 19. The method of claim 18, wherein the diabeticretinopathy is proliferative diabetic retinopathy.